Measles Virus
The invention relates to a vaccine containing recombinant attenuated measles viruses expressing antigens of Plasmodium falciparum (Pf) and to their use for the preparation of recombinant measles-malaria vaccine which will confer immunity against both Measles and Malaria antigens.
Measles virus (MV) is a member of the order Mononegavirales, i.e. viruses with a non-segmented negative-strand RNA genome. The non segmented genome of MV has an antimessage polarity; thus, the genomic RNA is not translated either in vivo or in vitro. Furthermore, it is biologically active only when it is very specifically associated with three viral proteins in the form of a ribonucleoprotein (RNP) complex (see below). Transcription and replication of non-segmented (−) strand RNA viruses and their assembly as virus particles have been reviewed extensively (1). Transcription and replication of measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of infected cells. The genome of the measles virus comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and additionally two-non structural proteins derived from the P gene, C and V, involved in counteracting the constitutive immune responses and in regulation of transcription/replication. The gene order is 3′ N, P (including C and V), M, F, H, and L 5′. In addition, from the 3′-terminal region a short leader RNA of about 50 nucleotides is transcribed. The cited genes respectively encode the proteins of the ribonucleocapsid (RNP) of the virus, i.e., the nucleoprotein (N), the phosphoprotein (P), and the large polymerase/replicase protein (L), which very tightly associate with the genome RNA, forming the RNP. The other genes encode the proteins of the viral envelope including the hemagglutinin (H), the fusion (F) and the matrix (M) proteins. The transcription of the MV genes follows a decreasing gradient: when the polymerase operates on the genomic template it synthesizes more RNA made from upstream genes than from downstream genes. In this discontinuous transcription mode the mRNAs are capped and polyadenylated. Conversely, in the replication mode, the L protein produces full length antigenomic and genomic RNA which are immediately covered with N, P and L proteins to form infectious progeny RNPs.
The measles virus has been isolated in 1954: Enders and Peebles inoculated primary human kidney cells with the blood of David Edmoston, a child affected by measles, and the resulting Edmoston strain of MV (2) was subsequently adapted to growth in a variety of cell lines. Adaptation to chicken embryos, chick embryo fibroblasts (CEF), and/or dog kidney cells and human diploid cells produced the attenuated Edmonston A and B (3), Zagreb (EZ) and AIK-C seeds. Edmonston B was licensed in 1963 as the first MV vaccine. Further passages of Edmonston A and B on CEF produced the more attenuated Schwarz and Moraten viruses (3) whose sequences have recently been shown to be identical (4; 5). Because Edmonston B vaccine was reactogenic, it was abandoned in 1975 and replaced by the Schwarz/Moraten vaccine. Several other vaccine strains are also used: AIK-C, Schwarz F88, CAM70, TD97 in Japan, Leningrad-16 in Russia, and Edmonston Zagreb. The CAM70 and TD97 Chinese strains were not derived from Edmonston. Schwarz/Moraten and AIK-C vaccines are produced on CEF. Zagreb vaccine is produced on human diploid cells (WI-38). Today, the Schwarz/Moraten, AIK-C and EZ vaccines are commonly used (6), but in principle, any one of these attenuated vaccine strains, which are all of the one unique MV serotype, proven to be safe and to induce long-lasting immune responses, can be used for the purposes of the invention.
MV vaccines induce life-long immunity after a single or two low-dose injections. Protection against measles is mediated both by antibodies and by CD4 and CD8 T cells. Persistence of MV-specific antibodies and CD8 cells has been shown for as long as 25 years after vaccination (7).
MV vaccine is easy to produce on a large scale in most countries and can be distributed at low cost. Because the attenuation of MV genome results from an advantageous combination of numerous mutations, the vaccine is very stable and reversion to pathogenicity has never been observed (6).
Regarding safety, MV replicates exclusively in the cytoplasm, ruling out the possibility of integration into host DNA. These characteristics make live attenuated MV vaccine an attractive candidate to be used as a multivalent vaccination vector. Such a vaccine may prove as efficient in eliciting long-lasting immune protection against other pathogenic agents as against the vector virus itself.
Martin Billeter and colleagues cloned cDNA corresponding to the antigenome of Edmonston MV, and established an original and efficient reverse genetics procedure to rescue the virus (8), as described in International Patent Application WO 97/06270. The recombinant measles virus is recovered from the helper cell line 293-3-46, stably transfected and expressing MV N an P proteins as well as bacteriophage T7 RNA polymerase. For rescue of any variant or recombinant MV the helper cell line is then transiently transfected with an expression plasmid encoding L protein, and most importantly with any antigenomic plasmid appropriately constructed to yield any mutated or recombinant antigenomic RNA compatible to give rise to progeny MV. The transient transfection step leads first to the transcription, preferably by the resident T7 RNA polymerase. The resulting antigenomic RNA is immediately (in statu nascendi) covered by the viral N, P and L proteins, to yield antigenomic RNP from which genomic RNP is produced. Second, the genomic RNP is transcribed by the attached L, to yield all viral mRNAs and the respective proteins. Finally, both genomic and antigenomic RNPs are amplified by replication.
In a slight variation of this procedure, rather than using stably transfected 293-3-46 helper cells, commercially available 293T cells have been transiently transfected, using simultaneously all 5 plasmids detailed in the original patent description, those encoding N, P and T7 polymerase (previously used to create the helper cell line) as well as the plasmid encoding L and the antigenomic plasmid. Note that in the “fully transient transfection” procedure it is possible to use also variant expression plasmids and to avoid the use of T7 RNA polymerase altogether, utilizing instead the resident RNA polymerase II to express also the L protein and the antigenome (9).
To rescue individual recombinant MVs the antigenomic plasmids utilized comprise the cDNA encoding the full length antigenomic (+)RNA of the measles virus recombined with nucleotide sequences encoding the heterologous antigen of interest (heterologous nucleotide sequence), flanked by MV-specific transcription start and termination sequences, thus forming additional transcription units (ATUs). This MV Edmonston strain vector has been developed by the original MV rescue inventors for the expression of foreign genes (10), demonstrating its large capacity of insertion (as much as 5 kb) and the high stability in the expression of transgenes (11; 12), such as Hepatitis B virus surface antigen, simian or human immunodeficiency viruses (SIV or HIV), mumps virus, and human IL-12. In particular, early on, recombinant measles virus expressing Hepatitis B virus surface and core antigens either individually or in combination have been produced and shown to induce humoral immune responses in genetically modified mice.
From the observation that the properties of the measles virus and especially its ability to elicit high titers of neutralizing antibodies in vivo and its property to be a potent inducer of long lasting cellular immune response, the inventors have proposed that it may be a good candidate for the production of recombinant viruses expressing antigens from P. falciparum, to induce neutralizing antibodies against said Malaria parasite which preferably could be suitable to achieve at least some degree of protection in animals and more preferably in human hosts.
Especially, MV strains and in particular vaccine strains have been elected in the present invention as candidate vectors to induce immunity against both measles virus and P. falciparum parasite whose constituent is expressed in the designed recombinant MV, in exposed infant populations because they are having no MV immunity.
Adult populations, even already MV immunized individuals, may however also benefit from MV recombinant immunization because re-administering MV virus under the recombinant form of the present invention results in a boost of anti-MV antibodies (13).
The invention relates in particular to the preparation of recombinant measles viruses bearing heterologous genes from P. falciparum parasites.
The advantageous immunological properties of the recombinant measles viruses according to the invention can be shown in an animal model which is chosen among animals susceptible to measles viruses, and wherein the humoral and/or cellular immune response against the heterologous antigen and/or against the measles virus is determined. Among such animals suitable to be used as model for the characterization of the immune response, the skilled person can especially use transgenic mice expressing CD46, one of the specific receptors for MV. The most promising recombinants can then be tested in monkeys.
The recombinant measles virus nucleotide sequence must comprise a total number of nucleotides which is a multiple of six. Adherence to this so-called “rule of six” is an absolute requirement not only for MV, but for all viruses belonging to the subfamily Paramyxovirinae. Apparently, the N protein molecules, each of which contacts six nucleotides, must cover the genomic and antigenomic RNAs precisely from the 5′ to the 3′ end.
It is of note that the location of the ATUs can vary along the antigenomic cDNA. Thus, taking advantage of the natural expression gradient of the mRNAs of MV mentioned above, the level of expression of inserted ATUs can be varied to appropriate levels. Preferred locations of ATUs are upstream of the L-gene, upstream from the M gene and upstream of the N gene, resulting in low, medium and strong expression, respectively, of heterologous proteins.
Malaria Parasite.
Malaria currently represents one of the most prevalent infectious diseases in the world, especially in tropical and subtropical areas. Per year, malaria infections lead to severe illnesses in hundreds of million individuals worldwide, killing between 1 and 3 million, primarily young infants in developing and emerging countries. The widespread occurrence and elevated incidence of malaria are a consequence of the widespread ban of DDT and the increasing numbers of drug-resistant parasites as well as insecticide-resistant parasite vectors. Other factors include environmental and climatic changes, civil disturbances, and increased mobility of populations.
Malaria is caused by the mosquito-borne hematoprotozoan parasites belonging to the genus Plasmodium from the phylum Apicomplexa. Four species of Plasmodium genus infect humans: P. malariae, responsible for Malaria quartana, P. vivax and P. ovale, both of which cause Malaria tertiana, and P. falciparum, the pathogen of Malaria tropica and responsible for almost all fatal infections. Many others cause disease in animals, such as P. yoelii and P. berghei in mice.
Malaria parasites have a life cycle consisting of several stages. Each stage is able to induce specific immune responses directed against the corresponding occurring stage-specific antigens. Malaria parasites are transmitted to man by several species of female Anopheles mosquitoes. Infected mosquitoes inject the “sporozoite” form of the malaria parasite into the mammalian bloodstream. Sporozoites remain for a few minutes in the circulation before invading hepatocytes. At this stage, the parasite is located in the extra-cellular environment and is exposed to antibody attack, mainly directed to the “circumsporozoite” (CS) protein, a major component of the sporozoite surface. Once in the liver, the parasites replicate and develop into so-called “schizonts.” These schizonts occur in a ratio of up to 20,000 per infected cell. During this intra-cellular stage of the parasite, main players of the host immune response are T-lymphocytes, especially CD8+ T-lymphocytes. After about one week of liver infection, thousands of so-called “merozoites” are released into the bloodstream. Apical membrane antigen 1 (AMA1) and merozoite surface protein 1 (MSP1) are both present on merozoites that emerge from infected liver cells: they are essential components of the asexual blood-stage merozoite, responsible for invasion of erythrocytes. Once they enter red blood cells, they become targets of antibody-mediated immune response and T-cell secreted cytokines. After invading erythrocytes, the merozoites undergo several stages of replication, giving rise to so-called “trophozoites” and to schizonts and merozoites, which can infect new red blood cells. A limited amount of trophozoites may evolve into “gametocytes,” which constitute the parasite's sexual stage. When susceptible mosquitoes ingest erythrocytes, gametocytes are released from the erythrocytes, resulting in several male gametocytes and one female gametocyte. The fertilization of these gametes leads to zygote formation and subsequent transformation into ookinetes, then into oocysts, and finally into salivary gland sporozoites. Targeting antibodies against gametocyte stage-specific surface antigens can block this cycle within the mosquito mid gut. Such antibodies will not protect the mammalian host but will reduce malaria transmission by decreasing the number of infected mosquitoes and their parasite load.
The MSP-1 is synthesised as 190-200 kDa (d-190) precursor which is proteolytically processed into fragments of 83, 30, 38 and 42 kDa (d-42) during schizogony (14). At the time of erythrocytic invasion the 42-kDa is further cleaved to yield a 33 kDa fragment which is shed with the rest of the complex, and a 19 kDa fragment, which contains two epidermal growth factor (EGF)-like domains, that remains associated with the merozoite membrane during invasion. This secondary cleavage is a pre-requisite for successfully erythrocyte invasion (15).
MSP-1 is an essentially dimorphic protein exhibiting high conservation within the dimorphic alleles characterised by the K1 and MAD20 prototypes.
AMA-1 (16) is a structurally conserved type I integral membrane protein, comprising 622 aa in P. falciparum (PfAMA-1), organised in a cytosolic region (50 aa), a transmembrane region, and an ectodomain, which folds as an a N-terminal pro-sequence and three domains (DI, DII, DIII) Expression of the protein is maximal in late schizogony: the precursor of AMA-1 (83 kDa) is processed proteolytically, to cleave away the pro-sequence, converting the protein into a 66 kDa form, which allows the merozoite relocalisation. Antibodies recognise mainly DI and DII, and appear to react equally well with several allelic variants. Antibody responses to DIII are generally low, levels increasing in adults (17, 18).
PfAMA-1 contains 64 polymorphic positions (9 in the pro-sequence, 52 in the ectodomain, 3 in the cytosolic region), most of them are dimorphic, which are important epitopes for host immune responses. To develop PfAMA-1-based vaccines it should be important to cover the polymorphisms: Diversity Covering (DiCo1, 2 and 3) PfAMA-1 are artificial sequences representing, to the greatest extent possible, the naturally occurring polymorphism of the PfAMA1 ectodomain. It has been shown that they induce immune responses which are functional against a range of parasites carrying diverse PfAMA1 alleles. This approach may offer a means by which vaccines targeting PfAMA1 can be produced such that a strong and a functional protection against the broad range of naturally occurring PfAMA1 alleles can be induced. (19).
The CS protein (CSP) has about 420 aa and a molecular weight of 58 kDa. It represents the major surface protein of sporozoites: its function is fundamental for the maturation of sporozoites from oocystis and for the invasion of hepatocytes, which is mediated from a conserved motif of positively charged aminoacids. CSP is organised into two non-repetitive regions at 5′ and 3′ ends, and a variable species-specific central region, consisting of multiple repeats of four-residues-long motifs, which represents the main epitope within the CSP. Since CSP continues to be detectable for at least the first 3 days of schizogony, it is considered an attractive vaccine target for both antibody-mediated immuno response, directed against extracellular sporozoites, and cell-mediated immuno responses, directed against schizonts (20).
Current approaches to malaria vaccine development can be classified according to the different stages in which the parasite can exist, as described above.
Three types of possible vaccines can be distinguished: i) pre-erythrocytic vaccines, which are directed against sporozoites and/or schizont-infected cells. These types of vaccines are primarily CS-based, and should ideally confer sterile immunity, mediated by humoral and cellular immune responses, preventing malaria infection; ii) asexual blood-stage vaccines, which are directed against merozoites-infected cells: MSP1 and AMA1 are leading malaria vaccine candidates, designed to minimize clinical severity. These vaccines should reduce morbidity and mortality and are meant to prevent the parasite from entering and/or developing in the erythrocytes; iii) transmission-blocking vaccines, which are designed to hamper the parasite development in the mosquito host. This type of vaccine should favour the reduction of population-wide malaria infection rates. Next to these vaccines, the feasibility of developing malaria vaccines that target multiple stages of the parasite life cycle is being pursued in so-called multi-component and/or multi-stage vaccines.
Today's global malaria vaccine portfolio looks promising with 47 new vaccine candidates, 31 in preclinical development, narrowing down to 16 in clinical trials. One of these, the RTS,S vaccine, being developed by GSK Biologicals and PATH-MVI, should enter final phase III clinical trials in 2008 (21). Other interesting vaccine candidates are those based on live recombinant viruses used as vector, such as Modified Vaccinia Ankara (MVA), as described in International Patent Application US2006127413, poxvirus (U.S. Pat. No. 6,214,353, AU7060294, AU1668197, WO9428930, and U.S. Pat. No. 5,756,101), adenovirus (US2007071726, US2005265974, US2007088156 and CA2507915), cold-adapted attenuated influenza virus, or based on yeasts, such as Pichia pastoris and Saccharomyces spp., or on bacterial expression systems, such as Salmonella spp. (U.S. Pat. No. 5,112,749) and Escherichia coli (EB0191748) (22).
Currently, no commercially available vaccine against malaria is available, although the development of vaccines against malaria has already been initiated more than 30 years ago. Many factors make malaria vaccine development difficult and challenging. First, the size and genetic complexity of the parasite mean that each infection presents thousands of antigens to the human immune system. Understanding which of these can be a useful target for vaccine development has been complicated, and to date at least 40 different promising antigens have been identified. Second, the parasite changes through several life stages even while in the human host, presenting, at each stage of the life cycle, a different subset of molecules to the immune system. Third, the parasite has evolved a series of strategies that allow it to confuse, hide, and misdirect the human immune system. Finally, it is possible to have multiple malaria infections of not only different species but also of different strains at the same time.
Hence the present invention fulfil the long felt need of prior art by providing combined measles-malaria vaccine containing different attenuated recombinant measles-malaria vectors comprising a heterologous nucleic acid encoding several Plasmodium falciparum antigens.